T lymphocytes play a pivotal role in directing the immune responses against foreign pathogens, damaged or transformed cells. However, when deregulated, T cells may mediate a number of pathological diseases such as autoimmune diseases (AD), including type-1 diabetes, rheumatoid arthritis, multiple sclerosis, etc., and may significantly affect the development and progression of inflammatory diseases such as cardiovascular disease and stroke. (Sospedra, M., and Martin, R. (2005) Annu. Rev. Immunol. 23(7): 683-747; Menge, T., Weber, M. S., Hemmer, B., Kieseier, B. C., von Budingen, H. C., Warnke, C., Zamvil, S. S., Boster, A., Khan, O., Hartung, H. P., and Stuve, O. (2008) Drugs 68(17): 2445-68; Cope A. P., Schulze-Koops H., and Aringer M. (2007) Clin Exp Rheumatol. 25(5 Suppl 46):S4-11; Yoon J W, and Jun H S. (2005) Am J Ther. 12(6):580-91; Skapenko A., Leipe J., Lipsky P. E., and Schulze-Koops H. (2005) Arthritis Res. Ther. 7(Suppl 2):S4-14). The immune system is also responsible for the destruction of organ transplants, the so called graft versus host disease (GVHD)(Jacobsohn D. A., and Vogelsang G. B. (2007) Orphanet J. Rare Dis. 4:2:35; Ferrara J. L., Cooke K. R., and Teshima T. (2003) Int. J. Hematol. 78(3):181-7). Currently available therapies for these T cell-mediated diseases are largely unsatisfactory primarily because all the available drugs are either only partially effective in reducing exacerbation and slowing down the disease progression, or produce severe side effects.
Naïve or memory T cells are quiescent cells circulating in the body and are commonly referred to as resting T cells. Resting T cells express T cell receptors (TCR) that recognize a specific antigen on an antigen-presenting cell. In order to participate in immune response, resting T cells undergo process of activation triggered by TCR crosslinking with an antigen (Favero, J. and Lafont, V (1998) Biochem. Pharmacol. 56(12): 1539-47; Killeen, N., Irving, B. A., Pippig, S., and Zingler, K. (1998) Curr. Opin. Immunol. 10(3): 360-7). During the activation phase, T cells start to express specific surface receptors and interleukins, and begin to proliferate with many rounds of divisions. In the presence of the repertoire of specific cytokines, activated T cells differentiate into effector T cells that participate in normal immune responses, such as responses directed against foreign pathogens (Swain, S. L. (1999) Curr. Opin. Immunol. 11(2): 180-5). When immune system function is impaired, activated T cells may direct immune responses against normal tissues, which causes AD, or against organ transplant, which causes GVHD. Auto-reactive activated T cells infiltrate sites of inflammation in patients with AD and are thought to drive the processes of tissue damage via secretion of pro-inflammatory cytokines, such as INF-γ, IL-17, IL-23, and TNF-α.
The process of T cell activation is tightly regulated. Calcium ions (Ca2+) play a critical role in T cell activation. In T lymphocytes, sustained elevation in intracellular calcium concentration ([Ca2+]i) induces a transcriptional program responsible for T cell clonal expansion and secretion of effector cytokines necessary for coordination of the immune response (Negulescu, P. A., Shastri, N., and Cahalan, M. D. (1994) Proceedings of the National Academy of Sciences of the United States of America 91(7), 2873-2877; Dolmetsch, R. E., Lewis, R. S., Goodnow, C. C., and Healy, J. I. (1997) Nature 386(6627), 855-858; Dolmetsch, R. E., Xu, K., and Lewis, R. S. (1998) Nature 392(6679), 933-936). Conversely, diminished [Ca2+]i signaling results in impaired T cell activation and, consequently, development of severe immunodeficiency (Gwack, Y., Feske, S., Srikanth, S., Hogan, P. G., and Rao, A. (2007) Cell Calcium 42(2):145-56; Feske, S. (2007) Nat Rev Immunol 7(9), 690-702).
TCR stimulation, by engagement with foreign antigen, for example, causes a biphasic increase in [Ca2+]i. The first increase results from Ca2+ release from intracellular stores. This is a consequence of inositol 1,4,5-trisphosphate (IP3) formation. IP3 binding by the IP3 receptor (IP3R) evokes Ca2+ release, predominantly from the endoplasmic recticulum. This first phase is followed by activation of the plasmalemmal store-operated Ca2+ (SOC) channels, which allows for the store-operated Ca2+ influx (SOCE) across the plasma membrane (Lewis, R. S. (2001) Annual Review of Immunology 19, 497-521; Putney, J. W., Jr. (1986) Cell Calcium 7(1), 1-12; Putney, J. W., Jr., and McKay, R. R. (1999) Bioessays 21(1), 38-46).
Recent studies have identified members of the ryanodine receptor (RyR) family as contributing to the IP3-independent component of Ca2+ signaling in some types of immune cells. RyR antagonists ruthenium red and dantrolene inhibit murine T cell proliferation and IL-2 production following TCR stimulation, whereas diminished expression of the type 3 RyR reduces TCR-dependent [Ca2+]i signaling in Jurkat T cells, a human leukemia cell line derived from a single person (Dupuis, G., and Bastin, B. (1988) J Leukoc Biol 43(3), 238-247; Guse, A. H., da Silva, C. P., Berg, I., Skapenko, A. L., Weber, K., Heyer, P., Hohenegger, M., Ashamu, G. A., Schulze-Koops, H., Potter, B. V., and Mayr, G. W. (1999) Nature 398(6722), 70-73; Schwarzmann, N., Kunerth, S., Weber, K., Mayr, G. W., and Guse, A. H. (2002) J Biol Chem 277(52), 50636-50642).
In Jurkat T cells TCR activation stimulates production of cyclic adenosine 5′-diphosphate-ribose (cADPR) and/or nicotinic acid adenine dinucleotide phosphate (NAADP), both of which are agonists of the RyR family (Guse, A. H., da Silva, C. P., Berg, I., Skapenko, A. L., Weber, K., Heyer, P., Hohenegger, M., Ashamu, G. A., Schulze-Koops, H., Potter, B. V., and Mayr, G. W. (1999) Nature 398(6722), 70-73; Gasser, A., Glassmeier, G., Fliegert, R., Langhorst, M. F., Meinke, S., Hein, D., Kruger, S., Weber, K., Heiner, I., Oppenheimer, N., Schwarz, J. R., and Guse, A. H. (2006) J. Biol. Chem. 281(5), 2489-2496). Decreased cADPR levels also reduce the TCR-dependent [Ca2+]i signaling in Jurkat T cells (Guse, A. H., da Silva, C. P., Berg, I., Skapenko, A. L., Weber, K., Heyer, P., Hohenegger, M., Ashamu, G. A., Schulze-Koops, H., Potter, B. V., and Mayr, G. W. (1999) Nature 398(6722), 70-73; Schwarzmann, N., Kunerth, S., Weber, K., Mayr, G. W., and Guse, A. H. (2002) J. Biol. Chem. 277(52), 50636-50642). Mechanisms of activation, and the role of RyR in regulation of [Ca2+]i signaling and Ca2+-dependent functions in normal human T cells have not been previously elucidated. Recent studies described in this invention revealed for the first time that RyR are highly expressed in activated human T cells, where they are activated by SOCE and play essential role in controlling Ca2+ signaling and Ca2+-dependent functions of activated T lymphocytes.
There are three known isoforms of RyR: type 1 (RyR1), type 2 (RyR2), and type 3 (RyR3). The RyR1 and RyR2 were originally found in the sarcoplasmic reticulum of skeletal and cardiac muscles, respectively. Ca2+ release from the sarcoplasmic reticulum through these receptors plays a central role in regulating the contraction of skeletal and cardiac muscle fibers. The RyR3 has been detected in specific regions of the brain, nonmuscle tissues, and skeletal muscle as well.